We have recently discovered a novel means of gene regulation whereby both protein-coding and non-coding RNAs are able to cross-talk through a new RNA language. In this new regulatory dimension, both protein-coding and non-coding RNAs (e.g. pseudogenes) can communicate with each other by competing for common microRNAs, hence acting as competing endogenous RNAs or ceRNAs. We have described several ceRNAs that regulate the tumor suppressor PTEN, thereby ascribing tumor suppressive functions to numerous cancer-unrelated mRNAs and non-coding RNAs. Moreover, we have developed a methodology to computationally predict ceRNAs for cancer-associated genes based upon the identity of their microRNA response elements (MREs), which are the foundation of this new language. This allows for the prediction and identification of novel putative tumor suppressors and oncogenes, and, critically, for the functionalization of the entire cancer-relevant transcriptome. We therefore propose to further characterize oncogenic and tumor suppressive ceRNA networks in vitro. Additionally, to explore the function of ceRNAs in basic biology in vivo, we have created a series of mouse models that express pseudogenes or 3'UTRs or lack established PTEN ceRNAs in an inducible manner. We will cross these mice to existing mouse models of cancer to characterize the contribution of ceRNAs to the malignant phenotype in vivo. We propose to address these outstanding questions pertaining to this new transformative biological dimension with the following specific aims: (1) to identify and functionally characteriz novel tumor suppressive PTEN ceRNAs as well as to analyze mouse models lacking bona fide PTEN ceRNAs such as ZEB2 and VHL; (2) to explore the effect of PTEN 3'UTR overexpression on the malignant phenotype elicited by either PTEN heterozygosity or overexpression of the PTEN targeting miR106b~25 cluster; (3) to analyze the ceRNA activity of the pseudogene BRAFps towards its ancestral gene, the proto-oncogene BRAF, in vitro and in vivo and its contribution to cancer development. We strongly believe that our application is both highly innovative and timely, with the potential to significantly further our understanding of this novel process of gene regulation in cancer genetics and biology. PUBLIC HEALTH RELEVANCE: We have recently discovered a novel mode of gene regulation that is based on a new competitive endogenous RNA (ceRNA) language. Protein-coding and non-coding ceRNAs can cross talk by competing for microRNAs, which constitute the words of the ceRNA language. On the basis of this new theory, we use computational predictions and experimental validation to unravel extensive regulatory networks that impact the expression of prominent cancer genes, thus adding numerous novel players to the list of putative tumor suppressors and oncogenes. This application outlines an experimental strategy to further characterize, in vivo, the ceRNA biological dimension, with specific focus on the ceRNA networks of the critical tumor suppressor PTEN and the proto-oncogene BRAF.